The levels of observation that have been studied by the methodological developments of recent years in molecular biology, are the genes themselves, the translation of these genes into RNA, and the resulting proteins. The question of which gene is switched on at which point in the course of the development of an individual, and how the activation and inhibition of specific genes in specific cells and tissues are controlled is correlatable to the degree and character of the methylation of the genes or of the genome. In this respect, pathogenic conditions may manifest themselves in a changed methylation pattern of individual genes or of the genome.
DNA methylation plays a role, for example, in the regulation of the transcription, in genetic imprinting, and in tumorigenesis. Therefore, the identification of 5-methylcytosine as a component of genetic information is of considerable interest. However, 5-methylcytosine positions cannot be identified by sequencing since 5-methylcytosine has the same base pairing behaviour as cytosine. Moreover, the epigenetic information carried by 5-methylcytosine is completely lost during PCR amplification.
A relatively new and currently the most frequently used method for analysing DNA for 5-methylcytosine is based upon the specific reaction of bisulfite with cytosine which, upon subsequent alkaline hydrolysis, is converted to uracil which corresponds to thymidine in its base pairing behaviour. However, 5-methylcytosine remains unmodified under these conditions. Consequently, the original DNA is converted in such a manner that methylcytosine, which originally could not be distinguished from cytosine by its hybridisation behaviour, can now be detected as the only remaining cytosine using “normal” molecular biological techniques, for example, by amplification and hybridisation or sequencing. All of these techniques are based on base pairing which can now be fully exploited. In terms of sensitivity, the prior art is defined by a method which encloses the DNA to be analysed in an agarose matrix, thus preventing the diffusion and renaturation of the DNA (bisulfite only reacts with single-stranded DNA), and which replaces all precipitation and purification steps with fast dialysis (Olek A, Oswald J, Walter J. A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res. 1996 Dec. 15; 24(24):5064-6). Using this method, it is possible to analyse individual cells, which illustrates the potential of the method. However, currently only individual regions of a length of up to approximately 3000 base pairs are analysed, a global analysis of cells for thousands of possible methylation events is not possible. However, this method cannot reliably analyse very small fragments from small sample quantities either. These are lost through the matrix in spite of the diffusion protection.
An overview of the further known methods of detecting 5-methylcytosine may be gathered from the following review article: Rein, T., DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 1998, 26, 2255.
To date, barring few exceptions (e.g., Zeschnigk M, Lich C, Buiting K, Doerfler W, Horsthemke B. A single-tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus. Eur J Hum Genet. 1997 March-April; 5(2):94-8) the bisulfite technique is only used in research. Always, however, short, specific fragments of a known gene are amplified subsequent to a bisulfite treatment and either completely sequenced (Olek A, Walter J. The pre-implantation ontogeny of the H19 methylation imprint. Nat Genet. 1997 November; 17(3):275-6) or individual cytosine positions are detected by a primer extension reaction (Gonzalgo M L, Jones P A. Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res. 1997 Jun. 15; 25(12):2529-31, WO 95/00669) or by enzymatic digestion (Xiong Z, Laird P W. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 1997 Jun. 15; 25(12):2532-4). In addition, detection by hybridisation has also been described (Olek et al., WO 99/28498).
Further publications dealing with the use of the bisulfite technique for methylation detection in individual genes are: Grigg G, Clark S. Sequencing 5-methylcytosine residues in genomic DNA. Bioessays. 1994 June; 16(6):431-6, 431; Zeschnigk M, Schmitz B, Dittrich B, Buiting K, Horsthemke B, Doerfler W. Imprinted segments in the human genome: different DNA methylation patterns in the Prader-Willi/Angelman syndrome region as determined by the genomic sequencing method. Hum Mol Genet. 1997 March; 6(3):387-95; Feil R, Charlton J, Bird A P, Walter J, Reik W. Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing. Nucleic Acids Res. 1994 Feb. 25; 22(4):695-6; Martin V, Ribieras S, Song-Wang X, Rio M C, Dante R. Genomic sequencing indicates a correlation between DNA hypomethylation in the 5′ region of the pS2 gene and its expression in human breast cancer cell lines. Gene. 1995 May 19; 157(1-2):261-4; WO 97/46705, WO 95/15373, and WO 97/45560.
An overview of the Prior Art in oligomer array manufacturing can be gathered from a special edition of Nature Genetics (Nature Genetics Supplement, Volume 21, January 1999), published in January 1999, and from the literature cited therein.
Fluorescently labelled probes are often used for the scanning of immobilised DNA arrays. The simple attachment of Cy3 and Cy5 dyes to the 5′-OH of the specific probe are particularly suitable for fluorescence labels. The detection of the fluorescence of the hybridised probes may be carried out, for example via a confocal microscope. Cy3 and Cy5 dyes, besides many others, are commercially available.
Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI-TOF) is a very efficient development for the analysis of biomolecules (Karas M, Hillenkamp F. Laser desorption ionisation of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988 Oct. 15; 60(20):2299-301). An analyte is embedded in a light-absorbing matrix. The matrix is evaporated by a short laser pulse thus transporting the analyte molecule into the vapour phase in an unfragmented manner. The analyte is ionised by collisions with matrix molecules. An applied voltage accelerates the ions into a field-free flight tube. Due to their different masses, the ions are accelerated at different rates. Smaller ions reach the detector sooner than bigger ones.
MALDI-TOF spectrometry is excellently suited to the analysis of peptides and proteins. The analysis of nucleic acids is somewhat more difficult (Gut I G, Beck S. DNA and Matrix Assisted Laser Desorption Ionization Mass Spectrometry. Current Innovations and Future Trends. 1995, 1; 147-57). The sensitivity to nucleic acids is approximately 100 times worse than to peptides and decreases disproportionally with increasing fragment size. For nucleic acids having a multiply negatively charged backbone, the ionisation process via the matrix is considerably less efficient. In MALDI-TOF spectrometry, the selection of the matrix plays an eminently important role. For the desorption of peptides, several very efficient matrixes have been found which produce a very fine crystallisation. There are now several responsive matrixes for DNA, however, the difference in sensitivity has not been reduced. The difference in sensitivity can be reduced by chemically modifying the DNA in such a manner that it becomes more similar to a peptide. Phosphorothioate nucleic acids in which the usual phosphates of the backbone are substituted with thiophosphates can be converted into a charge-neutral DNA using simple alkylation chemistry (Gut I G, Beck S. A procedure for selective DNA alkylation and detection by mass spectrometry. Nucleic Acids Res. 1995 Apr. 25; 23(8): 1367-73). The coupling of a charge tag to this modified DNA results in an increase in sensitivity to the same level as that found for peptides. A further advantage of charge tagging is the increased stability of the analysis against impurities which make the detection of unmodified substrates considerably more difficult.
Genomic DNA is obtained from DNA of cell, tissue or other test samples using standard methods. This standard methodology is found in references such as Fritsch and Maniatis eds., Molecular Cloning: A Laboratory Manual, 1989.
Breast cancer is currently the second most common type of cancer amongst women. In 2001 over 190,000 new cases of invasive breast cancer and over 47,000 additional cases of in situ breast cancer were diagnosed. Incidence and death rates increase with age, for the period 1994-1998 the incidence of breast cancer amongst women between the ages of 20 and 24 was only 1.5 per 100,000 population. However the risk increases to 489.7 within the age group 75-79. Mortality rates have decreased by approximately 5% over the last decade and factors affecting 5 year survival rates include age, stage of cancer, socioeconomic factors and race.
Breast cancer is defined as the uncontrolled proliferation of cells within breasts tissues. Breasts are comprised of 15 to 20 lobes joined together by ducts. Cancer arises most commonly in the duct, but is also found in the lobes with the rarest type of cancer termed inflammatory breast cancer.
It will be appreciated by those skilled in the art that there exists a continuing need to improve methods of early detection, classification and treatment of breast cancers. In contrast to the detection of some other common cancers such as cervical and dermal there are inherent difficulties in classifying and detecting breast cancers.
The first step of any treatment is the assessment of the patient's condition comparative to defined classifications of the disease. However the value of such a system is inherently dependant upon the quality of the classification. Breast cancers are staged according to their size, location and occurrence of metastasis. Methods of treatment include the use of surgery, radiation therapy, chemotherapy and endocrine therapy, which are also used as adjuvant therapies to surgery.
Endocrine therapies have been developed in order to block the effects of estrogen on cancer cells or to reduce serum estrogen levels. Tamoxifen (TAM) is the most widely used antioestrogenic drug for breast cancer patients. It acts blocking estrogen stimulation of breast cancer cells, inhibiting both translocation and nuclear binding of estrogen receptor. TAM is used in adjuvant setting for primary breast cancer patients and for the treatment of metastatic disease and its effectiveness has been proved in several clinical trials. Treatment for five years reduces annual disease recurrence by 47% and annual deaths by 26% and this reduction is similar in different age groups. Also it reduces the incidence of developing contralateral breast tumours. TAM is among the least toxic antineoplastic agents but as it has estrogenic properties in some tissues, it increases by 3 folds the risk of endometrial cancer. Other endocrine therapies include aromatase inhibitors, which block the enzyme aromatase in adipose tissue (the main source of estrogen in postmenopausal women) and lead to reduced or abolished production of estrogens in adipose tissue. Also, new agents which block or modulate the estrogen receptor have been developed, they are generally summarised as SERMs=selective estrogen receptor modulators. Yet another way to reduce the amount of estrogen available to the tumour is to apply agents which interrupt the feed-back loop in sex hormone regulation (LH-RH analogues) at a higher level.
In general, the side-effects of endocrine treatment are harmless compared to chemotherapy. However, as endocrine treatments eliminate or reduce estrogen as a growth factor for cancer, they work only for tumours which rely on estrogen as a growth factor. The growth of cancers which do not have a functional ER pathway cannot be modulated by endocrine therapies.
The current way to determine if a tumour will respond to endocrine therapies is the analysis of expression of hormone receptors. Hormone receptor status should be tested prior to any endocrine treatment as only a group of patients will benefit from therapy. For this purpose ER (estrogen receptor) and PR (progesterone receptor) status are tested routinely in all patients and they are considered predictive markers of response (a predictive marker or factor is any measurement associated with response or lack of response to a particular therapy). ER status is predictive of response in adjuvant endocrine therapy setting and also in advanced metastatic disease.
Currently, ER positive and or PR positive patients receive TAM and the remaining 10% of patients receive chemotherapy. The problem is that the endocrine treatment is only effective in a subgroup of hormone receptor positive patients and also, that a small subgroup of ER negative patients appear initially to respond to TAM. Then, the non responder subgroup will not only not benefit from endocrine treatment but have the adverse effects of it and will not have the opportunity to receive chemotherapy instead. On the other hand, the ER negative subgroup that could have benefit from endocrine treatment will receive instead chemotherapy. Several different assays are available to measure PR and ER, including biochemical and IHC (immunohistochemical) analysis, but there is a lack of standardisation of staining methods and interlaboratory variability (Clin Cancer Res 2000, 6:616-621).
Currently several predictive markers are under evaluation. As up to now most patients have received Tamoxifen as endocrine treatment most of the markers have been shown to be associated with response or resistance to tamoxifen. However, it is generally assumed that there is a large overlap between responders to one or the other endocrine treatment. In fact, ER and PR expression are used to select patients for any endocrine treatment. Among the markers which have been associated with TAM response is bcl-2. High bcl-2 levels showed promising correlation to TAM therapy response in patients with metastatic disease and prolonged survival and added valuable information to ER negative patient subgroup (J Clin Oncology 1997, 15 5:1916-1922; Endocrine, 2000, 13(1):1-10). There is conflicting evidence regarding the independent predictive value of c-erbB2 (Her2/neu) over expression in patients with advance breast cancer that require further evaluation and verification (British J of Cancer 1999, 79 (7/8):1220-1226; J Natl Cancer Inst, 1998, 90 (21): 1601-1608).
Other predictive markers include SRC-1 (steroid receptor coactivator-1), CGA gene over expression, cell kinetics and S phase fraction assays (Breast Cancer Res and Treat, 1998, 48:87-92; Oncogene 2001, 20:6955-6959). Recently, uPA (Urokinase-type plasminogen activator) and PAI-1 (Plasminogen activator inhibitor type 1) together showed to be useful to define a subgroup of patients who have worse prognosis and who would benefit from adjuvant systemic therapy (J Clinical Oncology, 2002, 20 n° 4). All of these markers need further evaluations in prospective trials as none of them is yet a validated marker of response.
A number of cancer-associated genes have been shown to be inactivated by hypermethylation of CpG islands during breast tumorigenesis. Decreased expression of the calcium binding protein S100A2 (Accession number NM—005978) has been associated with the development of breast cancers. Hypermethylation of the promoter region of this gene has been observed in neoplastic cells thus providing evidence that S100A2 repression in tumour cells is mediated by site-specific methylation.
The SYK gene (Accession number NM—003177) encodes a protein tyrosine kinase, Syk (spleen tyrosine kinase), that is highly expressed in hematopoietic cells. Syk is expressed in normal breast ductal epithelial cells but not in a subset of invasive breast carcinoma. Also, the loss of Syk expression seems to be associated with malignant phenotypes such as increased motility and invasion. The loss of expression occurs at the transcriptional level, and, as indicated by Yuan Y, Mendez R, Sahin A and Dai J L (Hypermethylation leads to silencing of the SYK gene in human breast cancer. Cancer Res. 2001 Jul. 15; 61(14):5558-61.), as a result of DNA hypermethylation.
The TGF-β type 2 receptor (encoded by the TGFBR2 gene, NM—003242) plays a role in trans-membrane signalling pathways via a complex of serine/threonine kinases. Mutations in the gene have been detected in some primary tumours and in several types of tumour-derived cell lines, including breast (Lucke C D, Philpott A, Metcalfe J C, Thompson A M, Hughes-Davies L, Kemp P R, Hesketh R. ‘Inhibiting mutations in the transforming growth factor beta type 2 receptor in recurrent human breast cancer.’ Cancer Res. 2001 Jan. 15; 61(2):482-5.).
The genes COX7A2L and GRIN2D were both identified as novel estrogen responsive elements by Watanabe et. al. (Isolation of estrogen-responsive genes with a CpG island library. Molec. Cell. Biol. 18: 442-449, 1998.) using the CpG-GBS (genomic binding site) method. The gene COX7A2L (Accession number NM—004718 encodes a, polypeptide 2-like cytochrome C oxidase subunit VIIA. Northern blot analysis detected an upregulation of COX7A2L after estrogen treatment of a breast cancer cell line. The gene GRIN2D (Accession number NM—000836) encodes the N-methyl-D-aspartate, ionotropic, subunit 2D glutamate receptor, a subunit of the NMDA receptor channels associated with neuronal signalling, furthermore expression of the cDNA has been observed in an osteosarcoma cell line. The gene VTN (also known as Vitronectin Accession number NM—000638) encodes a 75-kD glycoprotein (also called serum spreading factor or complement S-protein) that promotes attachment and spreading of animal cells in vitro, inhibits cytolysis by the complement C5b-9 complex, and modulates antithrombin III-thrombin action in blood coagulation. Furthermore expression of this gene has been linked to progression and invasiveness of cancer cells.
The gene SFN (also known as Stratifin) encodes a polypeptide of the 14-3-3 family, 14-3-3 sigma. The 14-3-3 family of proteins mediates signal transduction by binding to phosphoserine-containing proteins. Expression of the SFN gene is lost in breast carcinomas, this is likely due to hypermethylatin during the early stages of neoplastic transformation (see Umbricht C B, Evron E, Gabrielson E, Ferguson A, Marks J, Sukumar S. Hypermethylation of 14-3-3 sigma (stratifin) is an early event in breast cancer. Oncogene. 2001 Jun. 7; 20(26):3348-53).
The gene PSA (Accession number NM—021154), also known as PSA-T, is not to be confused with the gene also popularly referred to as PSA (Accession number NM—001648) which encodes prostate specific antigen and whose technically correct name is kallikrein 3. The gene PSA-T encodes the protein phosphoserine aminotransferase which is the second step-catalysing enzyme in the serine biosynthesis pathway. Changes in gene expression levels have been monitored by mRNA expression analysis and upregulation of the gene has been identified in colonic carcinoma in a study of 6 samples (Electrophoresis 2002 June; 23(11):1667-76 mRNA differential display of gene expression in colonic carcinoma. Ojala P, Sundstrom J, Gronroos J M, Virtanen E, Talvinen K, Nevalainen T J.).
The gene stathimin (NM—005563) codes for an oncoprotein 18, also known as stathimin, a conserved cytosolic phosphoprotein that regulates microtubule dynamics. The protein is highly expressed in a variety of human malignancies. In human breast cancers the stahimin gene has shown to be up-regulated in a subset of the tumours.
The gene PRKCD encodes a member of the family of protein kinase c enzymes, and is involved in B cell signaling and in the regulation of growth, apoptosis, and differentiation of a variety of cell types.
Some of these molecules interact in a cascade-like manner. PRKCD activity that targets STMN1 is modulated by SFN binding and SYK phosphorylation. Together this influences tubulin polymerization that is required for cell division.
The gene MSMB (Accession number NM—002443) has been mapped to 10q1.2. It encodes the beta-microseminoprotein (MSP) which is one of the major proteins secreted by the prostate. Furthermore, it may be useful as a diagnostic marker for prostate cancer. Using mRNA analysis low levels of beta-MSP mRNA expression and protein have been linked to progression under endocrine therapy and it has been postulated that it may be indicative of potentially aggressive prostate cancer (see Sakai H, Tsurusaki T, Kanda S, Koji T, Xuan J W, Saito Y. ‘Prognostic significance of beta-microseminoprotein mRNA expression in prostate cancer.’ Prostate. 1999 Mar. 1; 38(4):278-84.).
The gene TP53 (Accession number NM—000546) encodes the protein p53, one of the most well characterised tumour suppressor proteins. The p53 protein acts as a transcription factor and serves as a key regulator of the cell cycle. Inactivation of this gene through mutation disrupts the cell cycle, which, in turn, assists in tumour formation. Methylation changes associated with this gene have been reported to be significant in breast cancer. Saraswati et. al. (Nature 405, 974-978 (22 Jun. 2000) ‘Compromised HOXA5 function can limit p53 expression in human breast tumours’ reported that low levels of p53 mRNA in breast tumours was correlated to methylation of the HOXA5 gene. The product of the HOX5A gene binds to the promoter region of the p53 and mediates expression of the gene. Methylation of the promoter region of the p53 gene itself has been reported (Kang J H, Kim S J, Noh D Y, Park I A, Choe K J, Yoo O J, Kang H S. ‘Methylation in the p53 promoter is a supplementary route to breast carcinogenesis: correlation between CpG methylation in the p53 promoter and the mutation of the p53 gene in the progression from ductal carcinoma in situ to invasive ductal carcinoma.’ Lab Invest. 2001 April; 81(4):573-9.). It was therein demonstrated that CpG methylation in the p53 promoter region is found in breast cancer and it was hypothesised that methylation in the p53 promoter region could be an alternative pathway to neoplastic progression in breast tumours. It has been observed that treatment with Tamoxifen decreases the level of expression of the p53 gene (Farczadi E, Kaszas I, Baki M, Szende B. ‘Changes in apoptosis, mitosis, Her2, p53 and Bcl2 expression in breast carcinomas after short-term tamoxifen treatment.’ Neoplasma. 2002; 49(2):101-3.)
The gene CYP2D6 (Accession number: NM—000106) is a member of the human cytochrome P450 (CYP) superfamily. Many members of this family are involved in drug metabolism (see for example Curr Drug Metab. 2002 June; 3(3):289-309. Rodrigues A D, Rushmore T H.), of these Cytochrome P450 CYP2D6 is one of the most extensively characterised. It is highly polymorphic (more than 70 variations of the gene have been described), and allelic variation can result in both increased and decreased enzymatic activity. The CYP2D6 enzyme catalyses the metabolism of a large number of clinically important drugs including antidepressants, neuroleptics, some antiarrhythmics (Nature 1990 Oct. 25; 347(6295):773-6 Identification of the primary gene defect at the cytochrome P450 CYP2D locus Gough A C, Miles J S, Spurr N K, Moss J E, Gaedigk A, Eichelbaum M, Wolf C R.).
The gene PTGS2 (Accession number NM—000963) encodes an inducible isozyme of prostaglandin-endoperoxide synthase (prostaglandin-endoperoxide synthase 2). Aberrant methylation of this gene has been identified in lung carcinoimas (Cancer Epidemiol Biomarkers Prev 2002 March; 11(3):291-7 Hierarchical clustering of lung cancer cell lines using DNA methylation markers. Virmani A K, Tsou J A, Siegmund K D, Shen L Y, Long T I, Laird P W, Gazdar A F, Laird-Offringa I A.).
The gene CGA (Accession number NM—000735) encodes the alpha polypetptide of glycoprotein hormones. Further, it has been identified as an estrogen receptor alpha (ER alpha)-responsive gene and overexpression of the gene has been linked to ER positivity in breast tumours. Bieche et. al. examined mRNA levels of said gene in 125 ER alpha-positive post-menopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen therapy. Initial results indicated significant links between CGA gene overexpression and Scarff-Bloom-Richardson histopathological grade I+II and progesterone and estrogen receptor positivity, which suggested that CGA is a marker of low tumour aggressiveness (‘Identification of CGA as a Novel Estrogen Receptor-responsive Gene in Breast Cancer: An Outstanding Candidate Marker to predict the Response to Endocrine TherapyCancer Research’ 61, 1652-1658, Feb. 15, 2001. Ivan Bieche, Beatrice Parfait, Vivianne Le Doussal, Martine Olivi, Marie-Christine Rio, Rosette Lidereau and Michel Vidaud). Further mRNA expression analysis linked CGA expression levels to Tamoxifen response, it was postulated that when combined with analysis of the marker ERBB2 (a marker of poor response) the gene may be useful as a predictive marker of tamoxifen responsiveness in breast cancer (Oncogene 2001 Oct. 18; 20(47):6955-9 The CGA gene as new predictor of the response to endocrine therapy in ER alpha-positive postmenopausal breast cancer patients. Bieche I, Parfait B, Nogues C, Andrieu C, Vidaud D, Spyratos F, Lidereau R, Vidaud M.). The authors provided significant data associating the expression of the gene CGA with Tamoxifen treatment response. However, said analyses have all focused upon the analysis of relative levels of mRNA expression. This is not a methodology that is suitable for a medium or high throughput, nor is it a suitable basis for the development of a clinical assay.
The gene PITX-2 (NM—000325) encodes a transcription factor (PITX-2) which is known to be expressed during development of anterior structures such as the eye, teeth, and anterior pituitary. Although the expression of this gene is associated with cell differentiation and proliferation it has no heretofore recognised role in carcinogenesis or responsiveness to endocrine treatment. Furthermore, to date no known analysis of the methylation state of this gene has been reported.
RASSF1A (Ras association domain family 1A) gene is a candidate tumour suppressor gene at 3p21.3. The Ras GTPases are a superfamily of molecular switches that regulate cellular proliferation and apoptosis in response to extra-cellular signals. It is purported that RASSF1A is a tumour suppressor gene, and epigenetic alterations of this gene have been observed in a variety of cancers. Methylation of RASSF1A has been associated with poor prognosis in primary non-small cell lung cancer (Kim D H, Kim J S, Ji Y I, Shim Y M, Kim H, Han J, Park J., ‘Hypermethylation of RASSF1A promoter is associated with the age at starting smoking and a poor prognosis in primary non-small cell lung cancer.’ Cancer Res. 2003 Jul. 1; 63(13):3743-6.). It has also been associated with the development of pancreatic cancer (Kuzmin I, Liu L, Dammann R, Geil L, Stanbridge E J, Wilczynski S P, Lerman M I, Pfeifer G P. ‘Inactivation of RAS association domain family 1A gene in cervical carcinomas and the role of human papillomavirus infection.’ Cancer Res. 2003 Apr. 15; 63(8):1888-93.), as well as testicular tumours and prostate carcinoma amongst others. The application of the methylation of this gene as a cancer diagnostic marker has been described in U.S. Pat. No. 6,596,488, it does not however describe its application in the selection of appropriate treatments regimens for patients.
Also located within 3p21 is the Dystroglycan precursor gene (Dystrophin-associated glycoprotein 1) (NM—004393). Dystroglycan (DG, also known as DAG1) is an adhesion molecule comprising two subunits namely alpha-DG and beta-DG. The molecule is responsible for crucial interactions between extracellular matrix and cytoplasmatic compartment and it has been hypothesised that as such it may contribute to progression to metastatic disease. Decreased expression of this gene has been associated with correlated with higher tumour grade and stage in colon, prostate and breast tumours.
The onecut-2 transcription factor gene (NM—004852) is located at 18q21.31 is a homeodomain transcription factor regulator of liver gene expression in adults and during development.
The trefoil factor (TFF) 2 gene (NM—003225) is a member of the trefoil family of proteins. They are normally expressed at highest levels in the mucosa of the gastrointestinal tract, however they are often expressed ectopically in primary tumours of other tissues, including breast. The expression of TFF 1 is regulated by estrogen in estrogen-responsive breast cancer cells in culture, its expression is associated with that of the estrogen receptor and TFF1 is a marker of hormone responsiveness in tumours. TFF1 promoter methylation has been observed in non-expressing gastric carcinoma-derived cell lines and tissues.
TMEFF2 (NM—016192) encodes a transmembrane protein containing an epidermal growth factor (EGF)-like motif and two follistatin domains. It has been shown to be overexpressed in prostate and brain tissues and it has been suggested that this is an androgen-regulated gene exhibiting antiproliferative effects in prostate cancer cells.
Methylation of the gene ESR1 (NM—000125), encoding the estrogen receptor has been linked to several cancer types including lung, oesophageal, brain and colorectal. The estrogen receptor (ESR) is a ligand-activated transcription factor composed of several domains important for hormone binding, DNA binding, and activation of transcription.
The PCAF (NM—003884) gene encodes the p300/CBP-Associated Factor (PCAF). CBP and p300 are large nuclear proteins that bind to many sequence-specific factors involved in cell growth and/or differentiation. The p300/CBP associated factor displays in vivo binding activity with CBP and p300. The protein has histone acetyl transferase activity with core histones and nucleosome core particles, indicating that it plays a direct role in transcriptional regulation. p300/CBP associated factor also associates with NF-kappa-B p65. This protein has been shown to regulate expression of the gene p53 by acetylation of Lys320 in the C-terminal portion of p53.
The WBP11 (NM—016312) gene encodes a nuclear protein, which co-localises with mRNA splicing factors and intermediate filament-containing perinuclear networks. It contains two proline-rich regions that bind to the WW domain of Npw38, a nuclear protein, and thus this protein is also called Npw38-binding protein NpwBP.